The experiments described in this project are designed to provide insights into some of the detailed molecular mechanisms involved in regulation of transcription in metazoan cells. They all combine biochemical and genetic approaches to address specific outstanding issues in the field. The following specific aims are proposed. 1. Activation. Two cell lines, which will allow genetic analyses of the TFIIK components TBP and TAFII32, will be constructed and studied. The experiments will utilize the chicken cell line DT40, and employ a technology developed previously in which the endogenous alleles of each gene will be disrupted in the presence of tetracycline (tet)-repressible transgene encoding the appropriate protein. How in vivo depletion influences cell growth and (presumably) brings about cell death will be determined. The effects of depletion on transcription, generally and of specific genes, will be measured, and the basis of any gene-specific effects investigated. The consequences of depletion on TFIID structure and on accumulation of other general transcription factors will be determined. A genetic complementation assay will be used to study the species specificity of these factors, as well as the requirement and role of specific protein domains (such as the N terminus of TBP) in the various functions of the proteins. 2. Repression. Studies to elucidate the mechanism employed by the Drosophila repressor Even- skipped (Eve) will be continued. Efforts will be made to prepare large amounts of a minimal, active Eve derivative in a concentrated form so that the Eve-TBP complex can be reconstituted and crystalized and its structure determined by X-ray diffraction. Selected mutations in TBP will be examined for their effects on Eve binding, and transcriptionally active TBP mutants compromised in Eve binding will be used to reconstitute in vitro transcription and the ability of Eve to repress transcription determined. The recently discovered inhibitory effect of phosphorylation on Eve repression activity and TBP binding will be investigated further. The sites of phosphorylation by GSK-3/Shaggy and casein kinase II will be mapped and mutated and the effects of the mutations determined. The mechanism by which the phosphorylated inhibitory domain functions will be investigated. 3. Regulation of Dorsal. Studies on the activities and interactions of three genetically- defined factors, Toll, Tube and Pelle, that function to activate the intracytoplasmic Dorsal signaling cascade in Drosophila will be continued. The unexpected ability of Tube to function in the nucleus will be pursued using in vitro assays, and the requirement for Pelle- catalyzed phosphorylation of the Tube death domain investigated. The mechanism by which Pelle autophosphorylation is regulated will be investigated, and the hypothesis that Pelle autophosphorylation is controled in the early embryo will be tested. Phosphorylation-dependent interactions involving the intracytoplasmic domain of the Toll receptor and Pelle will be studied to determine how Toll modulates Pelle kinase activity. Pelle phosphorylation sites in Pelle itself and Tube, as well as newly described site(s) in the Toll I1-1R homology region, will be mapped and the effects of mutations in functional assays determined.